Non-heme-iron containing oxygenases have been shown to be involved in a large number of metabolic processes in both mammalian and bacterial systems. The broad objective of this proposed research program is to continue our analysis of the involvement of non-heme iron in the catalytic pathway of bacterial dioxygenases, and to initiate comparative studies with the mammalian non-heme iron-containing monooxygenase phenylalanine hydroxylase. In these studies, we will utilize (1) Physical techniques such as resonance Raman, and EXAFS; (2) Chemical techniques such as the design of transition state analogs and other active-site directed ligands, metal replacement and chemical modification; and (3) Rapid reaction kinetic techniques.